Hemoregulatory compounds

ABSTRACT

The present invention relates to novel compounds which have hemoregulatory activities and can be used to stimulate hematopoiesis and for the treatment of viral, fungal and bacterial infectious diseases.

This application is a 371 of PCT/US96/18251, filed Nov. 12, 1996 whichclaims priority of Provisional Application No. 60/006,640, filed Nov.13, 1996.

FIELD OF THE INVENTION

The present invention relates to novel compounds which havehemoregulatory activities and can be used to stimulate hematopoiesis andfor the treatment of viral, fungal and bacterial infectious diseases.

BACKGROUND OF THE INVENTION

The hematopoietic system is a life-long cell renewal process whereby adefined stem cell population gives rise to a larger population ofmature, differentiated blood cells (Dexter TM. Stem cells in normalgrowth and disease. Br Med J 1987; 195:1192-1194) of at least ninedifferent cell lineages (erythrocytes, platelets, eosinophils,basophils, neutrophils, monocytes/macrophages, osteoclasts, andlymphocytes) (Metcalf D. The Molecular Control of Blood Cells. 1988;Harvard University Press, Cambridge, MA). Stem cells are also ultimatelyresponsible for regenerating bone marrow following treatment withcytotoxic agents or following bone marrow transplantation.

The major dose-limiting toxicities of most standard anti-neoplasticdrugs are related to bone marrow suppression, which if severe andprolonged, can give rise to life-threatening infectious and hemorrhagiccomplications. Myelosuppression is predictable and has been reported tobe dose-limiting in greater than 50% of single-agent Phase I trialscytotoxic compounds (Merrouche Y, Catimel G, Clavel M. Hematopoieticgrowth factors and chemoprotectants; should we move toward a two-stepprocess for phase I clinical trials in oncology? Ann Oncol 1993;4:471-474). The risk of infection is directly related to the degree ofmyelosuppression as measured by the severity and duration of neutropenia(Brody GP, Buckley M, Sathe YS, Freireich EJ. Quantitative relationshipbetween circulating leukocytes and infections with acute leukemia. AnnIn Med 1965; 64:328-334).

The control of hematopoiesis involves the interplay of a variety ofcytokines and growth factors during various stages of the hematopoieticcascade, including early pluripotent stem cells and mature circulatingeffector cells. These regulatory molecules include granulocyte colonystimulating factor (G-CSF), granulocyte-macrophage stimulating factor(GM-CSF), macrophage-colony stimulating factor (M-CSF), and a variety ofinterleukins which have overlapping, additive and synergistic actionswhich play major roles in host defence. Mechanistically, this isaccomplished by enhancing the production of granulocytes andmacrophages, as well as by the activation of effector cell functions(Moore MAS. Hemopoietic growth factor interactions: in vitro and in vivopreclinical evaluation. Cancer Surveys 1990; 9:7-80). These coordinatedactivities support optimal host defences which are necessary forfighting bacterial, viral and fungal infections.

Strategies to prevent or reduce the severity of neutropenia andmyelotoxicity include the use of hematopoietic growth factors and/orother hematopoietic cytokines. Such treatments are becoming commonpractice, in that they offer the potential of increased doses ofcytotoxic agents that may improve the therapeutic efficacy ofantineoplastic agents, and reduce the morbidity associated with theiruse (Steward WP. Granulocyte and granulocyte-macrophage colonystimulating factors, Lancet 1993; 342:153-157). Clinical studies havedemonstrated the G-, GM- and/or M-CSF may reduce the duration ofneutropenia, accelerate myeloid recovery, and reduceneutropenia-associated infections and other infectious complications inpatients with malignancies who are receiving cytotoxic chemotherapy orin high infectious-risk patients following bone marrow transplantation(Steward WP. Granulocyte and granulocyte-macrophage colony stimulatingfactors, Lancet 1993; 342:153-157 and Munn DH, Cheung NKV. Preclinicaland clinical studies of macrophage colong-stimulating factor. SeminOncol 1992; 19:395-407).

Synthetic peptides have been reported to induce the synthesis andrelease of haematoporetic mediators, including m-CSF from bone marrowstromal elements see U.S. patent application Ser. No. 08/001,905.

We have now found certain novel non-peptide compounds which have astimulative effect on myelopoietic cells. They are useful in stimulatingmyelopoiesis in patients suffering from reduced myelopoietic activity,including bone marrow damage, agranulocytosis and aplastic anemiaincluding patients having depressed bone marrow function due toimmunosuppressive treatment to suppress tissue reactions i.e. in bonemarrow transplant surgery. They may also be used to promote more rapidregeneration of bone marrow after cytostatic chemotherapy and radiationtherapy for neoplastic and viral diseases. They may be of particularvalue where patients have serious infections due to a lack of immuneresponse following bone marrow failure. They are useful in the treatmentand prevention of viral, fungal and bacterial disease.

SUMMARY OF THE INVENTION

This invention comprises compounds, hereinafter represented as Formula(I), which have hemoregulatory activities and can be used to stimulatehematopoiesis and in the prevention and treatment of bacterial, viraland fungal diseases.

These compounds are useful in the restoration of leukocytes in patientswith lowered cell counts resulting from a variety of clinicalsituations, such as surgical induced myelosuppression, AIDS, ARDS,congenital myelodysplacis, bone marrow and organ transplants; in theprotection of patients with leukopenia from infection; in the treatmentof severely burned patients and in the amelioration of themyelosuppression observed with some cell-cycle specific antiviral agentsand in the treatment of infections in patients who have had bone marrowtransplants, especially those with graft versus host disease, in thetreatment of tuberculosis and in the treatment of fevers of unknownorigin in humans and animals. The compounds are also useful in thetreatment and prevention of viral, fungal and bacterial infectiousdiseases, particularly Candida and Herpes in both immunosuppressed and"normal" subjects. They are useful in the treatment of sepsis caused bygram negative and gram positive organisms.

These compounds may also be used in combination with the myelosuppresiveagents of co-pending U.S. application Ser. No. 07/799,465 and U.S. Pat.No. 4,499,081, incorporated by reference herein, to provide alternatingpeaks of high and low activity in bone marrow cells, thus augmenting thenatural circadian rhythm of hematopoiesis. In this way, cytostatictherapy can be given at periods of low bone marrow activity, thusreducing the risk of bone marrow damage, while regeneration will bepromoted by the succeeding peak of activity. This invention is also apharmaceutical composition, which comprises a compound of Formula (I)and a pharmaceutically acceptable carrier.

This invention further constitutes a method for stimulating themyelopoietic system of an animal, including humans, which comprisesadministering to an animal in need thereof, an effective amount of acompound of Formula (I).

This invention also constitutes a method for preventing and treatingviral, fungal and bacterial infections in immunosuppressed and normalanimals, including humans, which comprises administering to an animal inneed thereof, an effective amount of a compound of Formula (I).

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention are represented by structural Formula I##STR1## A¹ and A² are independently Z--(CH₂)_(k) --(NR²)_(y) --.

Z is independently a 4-10 membered mono- or bicyclic heterocyclic ringsystem containing up to four heteroatoms N, O, S in the ring in which atleast one heteroatom is N, and wherein the ring is substituted orunsubstituted by one or two C₁₋₄ alkyl, F, Cl, Br, I, C₁₋₄ alkoxy,(CH₂)_(m) R₄, oxo, oxime, O--C₁₋₄ alkyloxime, hydroxy, N(R₃)₂, acylaminoor aminoacyl groups, 8, 9, 10 membered monocyclic ring systems beingexcluded;

R¹ and R² are independently hydrogen, C₁₋₄ alkylC(O)R₄, C₁₋₄ alkyl or R₁and R₂ are benzyl which is optionally substituted by one or two C₁₋₄alkyl, C₁₋₄ alkoxy, F, Cl, I, Br, OH, or N(R₃)₂ ;

R₃ is independently hydrogen, C₁₋₄ alkyl, or benzyl;

R₄ is independently OR₃, N(R₃)₂ or SR₃ ; and

k is an integer from 0 to 4;

m is an integer from 1 to 3;

n is 1 or 2;

y is zero or one;

or a pharmaceutically acceptable salt thereof.

C₁₋₄ alkyl groups may be straight or branched.

The compounds of the present invention may contain one or moreasymmetric carbon atoms and may exist in racemic and optically activeform. All these compounds and diastereomers are contemplated to bewithin the scope of the present invention.

Z in the above Formula (I) denotes an optionally substituted pyrrolyl,isopyrrolyl, pyrazolyl, isoimidazolyl, triazolyl, iosxazolyl, oxazolyl,thiazolyl, isothiazolyl, oxadiazolyl, pyridinyl, pyridazinyl,pyrimidinyl, pyrazinyl, pyrrolidinyl, piperazinyl, triazinyl,morpholinyl, indolyl, indoleninyl, isobenzazolyl, pyrindinyl,ioindazolyl, indoxazinyl, benzoxazolyl, quinolinyl, isoquinolinyl,cinnolinyl, quinazolinyl, naphthyridinyl, pyridopyridinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, indolinyl,2-pyrrolidonyl, imidazolyl, imidazolidinyl, imidazolinyl, piperidyl,tetrazolyl, quinuclidinyl, azetidinyl, or purinyl;

Preferred compounds are those wherein Z is optionally substitutedpyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, quinolinyl,tetrahydroquinolinyl, azetidinyl, or pyrrolidinyl;

More preferred compounds are those wherein Z is optionally substituted2-pyridinyl, 2-pyrimidinyl, 2-pyrazinyl, 2-pyrrolidon-5-yl, orpyrrolidinyl.

Preferred compounds are N,N -Bis(picolinoyl)-3-aminopyrrolidine,N,N-Bis(picolinoyl)-4-aminopiperidine andN,N-Bis(picolinoyl)4-aminoperhydroazepine.

Methods of Preparation

Compounds of Formula (1) wherein A¹, A², m, n and R¹ are defined as inFormula (I) are prepared by methods analogous to those described inScheme 1. Appropriate diamines (such as 1 in Scheme 1) are bis-acylatedwith appropriate heterocyclic acids (such as 2 in Scheme 1) in asuitable polar aprotic solvent (such as pyridine) to give the finalproduct. Compounds 1 and 2 are prepared by methods known in the art.##STR2##

In order to use a compound of the Formula (I) or a pharmaceuticallyacceptable salt thereof for the treatment of humans and other mammals itis normally formulated in accordance with standard pharmaceuticalpractice as a pharmaceutical composition.

According to a still further feature of the present invention there areprovided pharmaceutical compositions comprising as active ingredient oneor more compounds of Formula (I) as herein before defined orphysiologically compatible salts thereof, in association with apharmaceutical carrier or excipient. The compositions according to theinvention may be presented for example, in a form suitable for oral,nasal, parenteral or rectal administration.

As used herein, the term "pharmaceutical" includes veterinaryapplications of the invention. These compounds may be encapsulated,tableted or prepared in an emulsion or syrup for oral administration.Pharmaceutically acceptable solid or liquid carriers may be added toenhance or stabilize the composition, or to facilitate preparation ofthe composition. Liquid carriers include syrup, peanut oil, olive oil,glycerin, saline and water. Solid carriers include starch, lactose,calcium sulfate dihydrate, terra alba, magnesium stearate or stearicacid, talc, pectin, acacia, agar or gelatin. The carrier may alsoinclude a sustained release material such a glyceryl monostearate orglyceryl distearate, alone or with a wax. The amount of solid carriervaries but, preferably will be between about 20 mg to about 1 g perdosage unit. The pharmaceutical preparations are made following theconventional techniques of pharmacy involving milling, mixing,granulating, and compressing, when necessary, for tablet forms; ormilling, mixing and filling for hard gelatin capsule forms. Capsulescontaining one or several active ingredients may be produced, forexample, by mixing the active ingredients with inert carriers, such aslactose or sorbitol, and filling the mixture into gelatin capsules. Whena liquid carrier is used, the preparation will be in the form of asyrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such aliquid formulation may be administered directly p.o. or filled into asoft gelatin capsule. Organ specific carrier systems may also be used.

Alternately pharmaceutical compositions of the compounds of thisinvention, or derivatives thereof, may be formulated as solutions oflyophilized powders for parenteral administration. Powders may bereconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. The liquid formulationis generally a buffered, isotonic, aqueous solution. Examples ofsuitable diluents are normal isotonic saline solution, standard 5%dextrose in water or buffered sodium or ammonium acetate solution. Suchformulation is especially suitable for parenteral administration, butmay also be used for oral administration and contained in a metered doseinhaler or nebulizer for insufflation. It may be desirable to addexcipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose,acacia, polyethylene glycol, mannitol, sodium chloride or sodiumcitrate.

For rectal administration, a pulverized powder of the compounds of thisinvention may be combined with excipients such as cocoa butter,glycerin, gelatin or polyethylene glycols and molded into a suppository.The pulverized powders may also be compounded with an oily preparation,gel, cream or emulsion, buffered or unbuffered, and administered througha transdermal patch.

Nasal sprays may be formulated similarly in aqueous solution and packedinto spray containers either with an aerosol propellant or provided withmeans for manual compression.

Dosage units containing the compounds of this invention preferablycontain 0.05-50 mg, for example 0.05-5 mg of the compound of formula (I)or salt thereof.

According to a still further feature of the present invention there isprovided a method of stimulation of myelopoiesis which comprisesadministering an effective amount of a pharmaceutical composition ashereinbefore defined to a subject.

No unacceptable toxicological effects are expected when compounds of theinvention are administered in accordance with the present invention.

The biological activity of the compounds of Formula (I) are demonstratedby the following tests.

Induction of Hematopoietic Synergistic Activity in Stromal Cells

The murine bone marrow derived stromal cell line, C6.4 is grown in 12well plates in RPMI 1640 with 10% FBS. Upon reaching confluence, theC6.4 cells are washed and the media exchanged with fresh RPMI 1640without FBS. Confluent cell layers of murine C6.4 cells are treated withcompound. Cell-free supernatants are collected 18 hours later.Supernatants are fractionated with a Centricon-30 molecular weightcut-off membrane. C6.4 cell hematopoietic synergistic factor (HSF)activity is measured in a murine CFU-C assay.

CFU-C Assay

Bone marrow cells are obtained from C57B1/6 female mice and suspended inRPMI 1640 with 10% FBS. Bone marrow cells (7.5E+4 cells/mL) are culturedwith sub optimal levels of CFU plus dilutions of test C6.4 cell 30K-Esupernatants from above in a standard murine soft agar CFU-C assay. Cellaggregates >50 cells are counted as colonies. The number of agarcolonies counted is proportional to the amount of HSF present within theC6.4 bone marrow stromal line supernatant.

Effector Cell Function Assay

Female C57B1 mice are administered test compound IP or PO daily for 8days. Resident peritoneal exudate cells (PEC) utilized ex vivo fromtreated or untreated mice are harvested with cold calcium andmagnesium-free DPBS supplemented with heparin and antibiotics within 2-4hours following the last injection. Adherent PEM populations areprepared by incubating standardized PEC suspensions in microtiter dishesfor 2 hours at 37° C. (5% CO₂) and removing nonadherent cells by washingthe wells with warm buffer.

The superoxide dismutase-inhibitable (SOD) superoxide released byeffector cells in response to a in vitro stimulation by phorbolmyristate acetate (PMA) (100-200 nM) or pre-opsonized (autologous sera)live C. albicans (E:T=1:10) are quantitated in a microtiterferricytochrome c reduction assay. The assay is performed in thepresence of 1% gelatin/HBSS and 80uM ferricytochrome c in a total volumeof 200 uL/well. The nmoles of cytochrome c reduced/well is calculatedfrom spectrophotometric readings (550 nm) taken following a 1 hourincubation at 37° C. (5% CO₂). The amount of SOD-inhibitable cytochromec reduced is determined by the inclusion of wells containing SOD (200U/well). Baseline superoxide release is determined in the absence ofstimuli. Experimental data are expressed as a percentage of the controlgroup.

The following examples are illustrative and are not limiting of thecompounds of this invention.

EXAMPLE 1 N,N'-bis(picolinoyl)-4-aminopiperidine

a) N-tert-Butoxycarbonyl-4-piperidone oxime

To a stirred suspension of hydroxylamine hydrochloride (0.84 g, 12.0mmol) in methanol (10 mL) at 0° C. was added solid Na₂ CO₃ (0.64 g, 6.0mmol). The mixture was stirred for ca. 5 min. N-t-Boc-4-piperidone (1.99g, 10.0 mmol) was added and the mixture was stirred for ca 2 h. Thereaction mixture was concentrated in vacuo to ca. half its originalvolume, then diluted with saturated NaHCO₃ (100 mL) and extracted withCHCl₃ (4×50 mL). The combined organic layers were dried over Na₂ SO₄,filtered, and concentrated in vacuo to yield 1.86 g (87%) of the desiredproduct as a tan solid. MS (ES+) m/z 215.0 [M+H]⁺.

b) N-tert-Butoxycarbonyl-4-aminopiperidine

To a solution of the crude compound of Example 1(a) (1.75 g, 8.20 mmol)in 2% ammonia/methanol (150 mL, v/v) was added Raney Nickel (ca. 5 g,50% suspension in H₂ O (w/w)). The suspension was hydrogenated at 52 psifor ca 18 h, then filtered through a Celite pad and washed several timeswith methanol. The combined filtrates were passed through a 0.45 micronmembrane filter, then concentrated in vacuo to yield 1.51 g (92%) of thedesired material as a blue oil. This material was used directly in thenext step without further purification. MS (ES+) m/z 201.2 [M+H]⁺.

c) N-tert-Butoxycarbonyl-4-(picolinamido)piperidine

To a stirred solution of the compound of Example 1(b) (1.51 g, 7.5 mmol)in pyridine (75 mL) was added picolinic acid (1.49 g, 12.1 mmol) and EDC(2.30 g, 12.0 mmol). After ca. 18 h, the mixture was concentrated invacuo to a viscous brown oil. This was added to a rapidly-stirredmixture of EtOAc (100 mL), H₂ O (100 mL) and sat'd NaCl (100 mL). Afterstirring for 2 h, the phases were separated and the aqueous layer wasextracted with fresh EtOAc (2×100 mL). The combined organic layers werewashed with H₂ O (100 mL), dried over Na₂ SO₄, filtered, andconcentrated in vacuo to a dark yellow-orange syrup. Purification byflash chromatography (2/1 EtOAc/hexane, silica gel) afforded 1.81 g(79%) of the desired product as a white solid. MS (ES+) m/z 306.2[M+H]⁺.

d) N,N'-Bis(picolinoyl)-4-aminopiperidine

To a stirred solution of the compound of Example 1(c) (0.61 g, 2.0 mmol)in CH₂ Cl₂ (10 mL) was added neat TFA (10 mL). The mixture was stirredat RT for 45 min then concentrated in vacuo and azeotroped with tolueneto yield a clear oil. This residue was dissolved in DMF (20 mL) andchilled in to 0° C. Diisopropylethylamine (1.1 mL, 6.3 mmol), HOBt (0.81g, 6.0 mmol), DMAP (0.25 g, 2.0 mmol), picolinic acid (0.74 g, 6.0 mmol)and EDC (1.16 g, 6.0 mmol) were added sequentially. The mixture wasstirred for 17 h, concentrated in vacuo to ca. half its original volume,then added to a rapidly-stirred mixture of EtOAc (100 mL), H₂ O (100 mL)and sat'd NaCl (100 mL). After stirring for 1 h, the phases wereseparated and the aqueous layer was extracted with fresh EtOAc (2×100mL). The combined organic layers were washed with H₂ O (50 mL), driedover Na₂ SO₄, filtered, and concentrated in vacuo to a dark oil whichsolidified upon standing. Purification by flash chromatography (5/95MeOH/CHCl₃, silica gel) afforded 0.39 g (63%) of the desired product asa white solid. MS (ES+) m/z 311.0 [M+H]⁺.

EXAMPLE 2

Formulations for pharmaceutical use incorporating compounds of thepresent invention can be prepared in various forms and with numerousexcipients. Examples of such formulations are given below.

    ______________________________________                                        Tablets/Ingredients    Per Tablet                                             ______________________________________                                        1.        Active ingredient                                                                              0.5    mg                                                    (Cpd of Form. I)                                                    2.        Corn Starch      20     mg                                          3.        Alginic acid     20     mg                                          4.        Sodium alginate  20     mg                                          5.        Mg stearate      1.3    mg                                          ______________________________________                                    

Procedure for Tablets:

Step 1 Blend ingredients No. 1, No. 2, No. 3 and No. 4 in a suitablemixer/blender.

Step 2 Add sufficient water portion-wise to the blend from Step 1 withcareful mixing after each addition. Such additions of water and mixinguntil the mass is of a consistency to permit its converion to wetgranules.

Step 3 The wet mass is converted to granules by passing it through anoscillating granulator using a No. 8 mesh (2.38 mm) screen.

Step 4 The wet granules are then dried in an oven at 140° F. (60° C.)until dry.

Step 5 The dry granules are lubricated with ingredient No. 5.

Step 6 The lubricated granules are compressed on a suitable tabletpress.

Parenteral Formulation

A pharmaceutical composition for parenteral administration is preparedby dissolving an appropriate amount of a compound of Formula I inpolyethylene glycol with heating. This solution is then diluted withwater for injections Ph Eur. (to 100 ml). The solution is thensterilized by filtration through a 0.22 micron membrane filter andsealed in sterile containers.

What is claimed is:
 1. A compound of Formula (I): ##STR3## A¹ and A² areindependently Z; Z is independently 2-pyridyl, 2-pyrimidinyl,2-pyrazinyl, 2-pyrrolidon-5-yl or 2-pyrrolidinyl, which groups aresubstituted or unsubstituted by one or two C₁₋₄ alkyl, F, Cl, Br, I,C₁₋₄ alkoxy, (CH₂)_(m) R₄, oxo, oxime, O-C₁₋₄ alkyloxime, hydroxy,N(R₃)₂, acylamino or aminoacyl groups;R¹ is hydrogen, C₁₋₄ alkyl or R₁is benzyl which is optionally substituted by one or two C₁₋₄ alkyl, C₁₋₄alkoxy, F, Cl, I, Br, OH, or N(R₃)₂ ; R₃ is independently hydrogen, C₁₋₄alkyl, or benzyl; R₄ is independently OR₃, N(R₃)₂ or SR₃ ; and m is aninteger from 1 to 3; n is 1 or 2; or a pharmaceutically acceptable saltthereof.
 2. A compound of claim 1 selected from the group consisting ofN,N'-Bis(picolinoyl)-3-aminopyrrolidine,N,N'-Bis(picolinoyl)-4-aminopiperidine andN,N'-Bis(picolinoyl)-4-aminoperhydroazepine.
 3. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticalcarrier or excipient.
 4. A method of preventing or treating viral,fungal or bacterial infections which comprises administering to ananimal in need thereof, an effective amount of a compound of claim
 1. 5.A method of stimulating mylopoiesis in an animal in need thereof byadministering a compound of claim
 1. 6. A method of treating orpreventing sepsis in an animal in need thereof by administering acompound of claim 1.